A study in the Feb. 2, 2006 Nature by Antoine van Oijen’s lab sheds light on a longstanding puzzle in DNA replication: how do the enzymes that copy the two strands of DNA manage to coordinate their separate movements?
Replicating the genome is complicated by a quirk of DNA chemistry: DNA has polarity, and the two strands run like a two- lane highway in opposite directions. But DNA polymerase can only copy DNA in one direction. The two DNA polymerases chug along the two strands of DNA as a single complex. The polymerase on the leading strand can move without stopping, but the polymerase on the lagging strand must copy the DNA backwards in fragments. Each fragment requires the construction of a short RNA primer before replication can start again. Given these frequent delays, what keeps the lagging strand from getting hopelessly behind the leading strand? By taking advantage of the mechanical properties of DNA, van Oijen, Harvard Medical School assistant professor of biological chemistry and molecular pharmacology, was able to track the pace of these reactions in real time on the level of single molecules.
His team, including first author and research fellow Jong-Bong Lee, anchored one end of a piece of DNA to the inner surface of a glass flow cell; on the other end, they affixed a latex bead. When they allowed fluid to flow across the DNA at a constant speed, the bead would drag the DNA in the direction of the current, stretching it out to a fixed length. When DNA becomes single stranded, it coils up, making the length of the entire strand shorter. The researchers were able to use the position of the beads as an indicator of how much DNA was single- versus double-stranded, and thereby piece together the events taking place on both strands.
Using the pared-down replication machinery of a bacteriophage, the researchers found that during replication, the polymerase on the leading strand does not simply chug along at a constant pace. Instead, it pauses to wait whenever a primer is formed on the lagging strand. “It stops to allow the slow process of making this primer to take place,” said van Oijen. Further studies showed that the primase, the enzyme that constructs primers on the lagging strand, also acts as the brake keeping the polymerase on the leading strand from zooming ahead.